Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy

Abstract

A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

1. A plasmid comprising a heterologous pir gene having at least one mutation in the pir gene DNA binding region, wherein the at least one mutation is chosen from the 114C mutation, the 100B mutation, and the 201C mutation, and wherein the heterologous pir gene having at least one mutation in the pir gene DNA binding region encodes a π protein that activates a conditional origin of replication. 
     
     
       2. The plasmid of  claim 1 , further comprising at least one mutation in the pir gene leucine zipper-like motif. 
     
     
       3. The plasmid of  claim 1 , further comprising a mutation in the pir gene copy number control region. 
     
     
       4. The plasmid of  claim 1 , wherein the conditional origin of replication is from bacterial plasmid R6K. 
     
     
       5. The plasmid of  claim 1 , wherein the mutation in the DNA binding region is the 114C mutation. 
     
     
       6. The plasmid of  claim 1 , wherein the mutation in the DNA binding region is the 100B mutation. 
     
     
       7. The plasmid of  claim 1 , wherein the mutation in the DNA binding region is the 201C mutation. 
     
     
       8. A prokaryotic recombinant host cell comprising a heterologous pir gene and a DNA molecule comprising a heterologous gene encoding a protein, a selection gene, a target region for a site-specific recombinase, and a conditional origin of replication whose functionality in a prokaryotic host cell requires a π protein, wherein the heterologous pir gene comprises at least one mutation in the pir gene DNA binding region, wherein the at least one mutation is chosen from the 114C mutation, the 100B mutation, and the 201C mutation, and wherein the heterologous pir gene having at least one mutation in the pir gene DNA binding region encodes a π protein that activates the conditional origin of replication. 
     
     
       9. The prokaryotic recombinant host cell of  claim 8 , wherein the heterologous pir gene further comprises at least one mutation in the leucine zipper-like motif. 
     
     
       10. The prokaryotic recombinant host cell of  claim 8 , wherein the heterologous pir gene further comprises a mutation in the pir gene copy number control region. 
     
     
       11. The prokaryotic recombinant host cell of  claim 8 , wherein the heterologous pir gene is in a plasmid. 
     
     
       12. The prokaryotic recombinant host cell of  claim 8 , wherein the heterologous pir gene is in the genome of the host cell. 
     
     
       13. The prokaryotic recombinant host cell of  claim 8 , wherein the mutation in the DNA binding region is the 114C mutation. 
     
     
       14. The prokaryotic recombinant host cell of  claim 8 , wherein the mutation in the DNA binding region is the 100B mutation. 
     
     
       15. The prokaryotic recombinant host cell of  claim 8 , wherein the mutation in the DNA binding region is the 201C mutation. 
     
     
       16. The plasmid of  claim 3 , wherein the mutation in the copy number control region is pir 116. 
     
     
       17. The prokaryotic recombinant host cell of  claim 10 , wherein the mutation in the copy number control region is pir 116. 
     
     
       18. The prokaryotic recombinant host cell of  claim 8 , wherein the conditional origin of replication is from bacterial plasmid R6K. 
     
     
       19. The prokaryotic recombinant host cell according to  claim 8 , further comprising a gene comprising an amber mutation. 
     
     
       20. The prokaryotic recombinant host cell according to  claim 8 , wherein said host cell is deficient for the gene endA. 
     
     
       21. The prokaryotic recombinant host cell according to  claim 8 , wherein said host cell is deficient for the gene traD. 
     
     
       22. The prokaryotic recombinant host cell according to  claim 8 , wherein said host cell further comprises a mutated recA gene.